The Hedgehog (Hh) signal transduction pathway plays an essential role in patterning fields of cells during development, and is frequently activated in cancer. Inappropriate Hh signaling is causative in basal cell carcinoma, the most common cancer in humans, medulloblastoma, the most common malignant brain tumor in children, and rhabdomyosarcoma, where pathway activation correlates with poor prognosis. Hh has also been implicated in sporadic tumors of the lung, breast and prostate. Consequently, the Hh pathway has emerged as an attractive therapeutic target; specific inhibition of Hh signaling will likely have efficacy against these cancers without triggering the undesirable side effects associated with conventional chemotherapy. To design and employ Hh-specific therapies, a clear understanding of the regulatory mechanisms and signaling components controlling physiological Hh signal transduction is of paramount importance. Accordingly, the goals of this proposal are to dissect mechanistic events governing Hh pathway induction and to identify novel effectors facilitating downstream signaling. We will achieve these goals through molecular and functional analysis of Hh signal transduction using the fruit fly Drosophila melanogaster as our model system. Our studies primarily focus on elucidating the signaling mechanisms and downstream effectors of Smoothened, the critical signal transducer of the Hh pathway. Smoothened is a member of the G-protein coupled receptor (GPCR) superfamily. As such, this study includes three Specific Aims designed to better characterize Smoothened GPCR signaling activity: 1) To characterize the extracellular GPCR structural features that facilitate Hh- regulated Smoothened signaling, 2) To identify GPCR functional domains in Smoothened that govern signaling and 3) To map the G-protein effector network functioning downstream of Smoothened. The proposed studies will enhance our understanding of how the Hh signal is initiated and transduced, and may reveal novel therapeutic targets through identifying pressure points on Smoothened and within the Hh cascade.